Dennis S. Huang

In vivo and in vitro cocaine modulation on production of cytokines in C57BL/6 mice.

In the present study we used both in vivo and in vitro murine models to investigate the effects of cocaine on the release of cytokines (IL-1 alpha, IL-6, and TNF-alpha by peritoneal macrophages and splenocytes, IL-2, IL-4, IL-5, IL-10, and IFN-gamma produced by splenocytes. In vitro cocaine (0.1, 1, 10, 100 micrograms/ml) exposure inhibited all cytokines produced by ConA-stimulated spleen cells and LPS-stimulated macrophages in a concentration dependent manner. Different effects of cocaine administration on cytokine production were observed when female C57BL/6 mice were injected intraperitoneally with cocaine (40 mg/kg body weight for six weeks). Secretion of IL-2 by splenocytes was significantly enhanced by cocaine administration, whereas IFN-gamma was not affected. Secretion of IL-4 and IL-10 by splenocytes was significantly inhibited by cocaine administration, while secretion of IL-5 by splenocytes was significantly enhanced. Secretion of IL-6 and TNF-alpha by splenocytes was significantly enhanced by cocaine administration. Secretion of IL-6 by peritoneal macrophages was also significantly enhanced by the cocaine, while production of IL-1 alpha was not affected. However, release of TNF-alpha by peritoneal macrophages was significantly reduced by the cocaine administration. Therefore use of cocaine may alter the balance of cytokine production, and thereby adversely affects immune response and host defense.

Production of tumor necrosis factor α by resident and activated murine macrophages

Alveolar macrophages (Am⊘s), resident peritoneal macrophages (RPm⊘s), and thioglycolate‐elicited peritoneal macrophages (TGPm⊘s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon γ (IFN‐γ) for 24 h. Tumor necrosis factor (TNF) in cell‐free supernatants was measured by enzyme‐linked immunosorbent assay. Anio⊘s incubated with 103 ng/ml LPS produced 50 times more TNF than RPm⊘s and 5 times more than TGPm⊘s, and LPS alone induced maximum TNF production by Am⊘s. Stimulated cell supernatant or recombinant IFN‐γ alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN‐γ had only a limited synergistic effect on TNF production by Am⊘s. However, both LPS and stimulated cell supernatant or recombinant IFN‐γ induced maximum TNF production by RPm⊘s and TGPm⊘s. TGPm⊘s showed greater sensitivity to LPS and stimulated cell supernatant or IFN‐γ with regard to TNF production than the other macrophage populations investigated.

Ethanol modulation of tumor necrosis factor and gamma interferon production by murine splenocytes and macrophages

C57BL/6 female mice were fed a liquid ethanol (ETOH) diet (27% of calories derived from ETOH) for 5 months as an animal model of chronic alcohol use. A isocaloric liquid diet supplemented with maltose dextran was fed to controls. Splenocytes from ETOH-treated and control mice, and purified macrophages from normal mice and retrovirus infected mice were exposed in vitro to various concentrations of ETOH (0.1-1.0% v/v). The effect of chronic ETOH exposure in vivo, and of in vitro treatment with ETOH on tumor necrosis factor (TNF) and gamma-interferon (IFN) production by cultured murine splenocytes and purified macrophages was investigated. Dietary ethanol did not significantly affect in vitro TNF or IFN production. However, TNF and IFN production by splenocytes from mice fed either the ETOH or control diet decreased significantly when the cells were cultured with ETOH and stimulated with LPS or Con A in vitro. Thus ETOH in vitro directly down regulates TNF and IFN secretion by LPS- or Con A-stimulated spleen cells. ETOH treatment in vitro did not significantly change TNF production by purified peritoneal macrophage (PM) and thioglycollate-induced peritoneal macrophage (TPM) from normal mice, but increased TNF production by alveolar macrophage (AM). Although murine retrovirus infection per se increased TNF production it did not change the responsive pattern in TNF production of PM and TPM to ETOH in vitro and reduced the TNF production of AM.

Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS

LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C57BL/6 female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LP-BM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CD8+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8+ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS.

Beta-carotene in vitro stimulates tumor necrosis factor alpha and interleukin 1 alpha secretion by human peripheral blood mononuclear cells

Beta-carotene (BC) potentially affects cancer resistance by stimulating secretion of immunoregulatory cytokines and thereby modulating immune defenses. Therefore, we investigated the effects of BC applied in vitro on the secretion of interleukin-1 alpha (IL-1), tumor necrosis factor alpha (IL-1), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were activated with pokeweed mitogen (PWM; 0.1 ug/ml), or lipopolysaccharide (LPS; 10 ug/ml) for 24 hr, or phytohemagglutinin (PHA; 5 ug/ml) for 74 hr. BC was encapsulated in liposomes and delivered to PBMC during mitogen activation at concentrations of 10−6 to 10−11M. The concentration of cytokines in the supernatants of activated cells was measured by ELISA. BC had no impact on IFN release. The release of IL-1 was significantly (p<0.05) stimulated by BC (10−6 to 10−8 M). No effect on IL-1 secretion was obtained when PBMC were incubated with BC-free liposomes. However, BC-free liposomes suppressed significantly TNF secretion (from 1.5 to 0.2 ng/ml). When BC was presented in liposomes at concentrations ranging from 10−6 to 10−8 M it stimulated significantly (p<0.01) TNF secretion. We suggest that physiologically achievable concentration so BC stimulate monokine secretion. Such changes might explain in part the observed immunomodulatory effect of BC on lymphoid cells and reduced cancer risk associated with high intake of BC.

Splenocyte subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine.

An experimental model which resembles human drug addiction was developed to study the effect of chronic drug (cocaine or morphine) administration on the immune system. As malnutrition has been associated with drug use, a low protein diet has been evaluated for its contribution to the impairment of the immune system during cocaine/morphine addiction. Female C57BL/6 mice that received a 20% or 4% casein diet were studied. Both drugs were administered intraperitoneally daily for 11 weeks and drugs were administered in increasing daily doses, beginning after 3 weeks of diet consumption. Doses of cocaine began with 5 mg/kg body weight and reached the maximum dose of 40 mg/kg/day at the fourth week. Doses of morphine gradually increased from 10 mg/kg to 75 mg/kg body weight with the maximum dose reached after 5 weeks of treatment. Cocaine administration reduced body weight, particularly in the low protein diet group, and spleen weight in protein malnourished mice. Cocaine as well as saline injected mice showed a decrease in the percentage of CD4+ CD8+ and Mac-1+ cells and an increase in B cells in the spleens of well nourished mice. Morphine-treated mice showed similar results to those observed in cocaine or saline treated mice. These results suggest that cocaine, morphine or saline injection can alter the percentage of cells that express a defined phenotype independently of the nutritional status of the subject. Moreover, the effect appears dependent on a stress mediated process.

Modification of spleen cell subsets by chronic cocaine administration and murine retrovirus infection in normal and protein-malnourished mice.

We have developed an experimental mouse model to study the effect of daily cocaine administration on the immune system during an acquired immune deficiency syndrome (AIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with the development of functional murine AIDS. Increasing doses of cocaine given by daily intraperitoneal injection for 11 weeks reduced body weight. A daily cocaine injection in some mice as well as a saline injection in others showed a decrease in the percentage of Thy 1.2+, CD4+ and CD8+ cells, while both treatments increased the percentage and absolute numbers of B-cells per spleen. Saline and cocaine treatment induced an increase in gamma-IFN and TNF-alpha production by splenocytes. Cocaine treatment favored a decrease in sIL-2R secretion. Saline and cocaine treatment had slightly different effects on the splenocytes of protein-malnourished mice. Cocaine treatment induced an increase in the percentage of CD8+ cells. Saline and cocaine treatments decreased the number of Mac 1+ cells in the spleen. Moreover, saline- and cocaine-treated protein-malnourished mice splenocytes did not present the increase in gamma-IFN production as well-nourished mice splenocytes showed. Retrovirus-infected mice showed a decrease in the percentage of Thy 1.2+ and CD8+ cells and an increase in the percentage and absolute numbers of CD4+, IL-2R+, Mac 1+ and B-cells. Cocaine partially prevented the enlargement of lymphoid organs due to lymphoid cell proliferation induced by murine retrovirus infection, but had little effect on the elevated percentage of CD4+ cells or B-cells or the depressed numbers of CD8+ cells associated with virus infection. However, cocaine did reduce the number of activated IL-2R+ cells and macrophages (Mac 1+) in addition to reducing the total number of cells per spleen in all subsets in retrovirus-infected mice, but not in uninfected controls. Cocaine treatment and retrovirus infection alone or in combination suppressed the release of sIL-2R into supernatant fluid during in vitro culture of splenocytes. These data illustrate that cocaine treatment modulates cell proliferation in retrovirus-infected mice and thus modifies the absolute number of cells in those subsets already altered by retrovirus infection. Retrovirus-infected and retrovirus-infected cocaine-treated protein-malnourished mice showed similar results.

Cocaine modulation in vitro of tumor necrosis factor production by macrophages from retrovirally infected mice

The effect of cocaine and LP-BM5 murine leukemia retrovirus infection on tumor necrosis factor (TNF) production were investigated. Three types of macrophages were used 1) peritoneal macrophage (PM), 2) thioglycollate induced peritoneal macrophage (TPM), and 3) alveolar macrophage (AM). Cells were cultured with and without cocaine during in vitro stimulation by lipopolysaccharide (LPS) and gamma-interferon (IFN). Retroviral infection enhanced the TNF production by PM and AM, but not by TPM. Intraperitoneal cocaine injection reduced TNF production by PM, but increased TNF production by AM and TPM. TNF production by AM from cocaine injected mice was stimulated by cocaine applied in vitro. In contrast, 100 micrograms/ml of cocaine in vitro significantly inhibited the TNF production by TPM from uninfected and retrovirally infected mice. Thus, TNF production by macrophages is modulated by murine retroviral infection and cocaine treatment. This could play an important role in host defense.

Spleen and thymus cell subsets modified by long-term morphine administration and murine AIDS--II.

Intravenous heroin abusers suffer a great variety of infections, including AIDS (acquired immune deficiency syndrome). We developed an experimental mouse model to evaluate the long-term effect of in vivo morphine administration during retrovirus-induced immune dysfunction. Mice were treated daily for 11 weeks with increasing doses of morphine. Morphine treatment produced a decrease in body weight and spleen cell number. Murine retrovirus infection provoked an increase in body weight due to enlargement of lymphoid organs, and an increase in the percentage and absolute number of CD4+ and Mac 1+ cells. Interestingly, retrovirus-infected mice that were also morphine-treated did not show the increase in the relative proportion of Mac 1+ cells. Moreover, under the experimental conditions of protein-malnutrition and morphine treatment potentiation of immune dysfunction by murine retrovirus infection was investigated. Retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. Splenocytes from retrovirus-infected mice presented a higher percentage of IL-2R+ cells and, lower levels of sIL-2R in splenocyte supernatants. Mitogen-stimulated splenocytes had a lower production of interferon-gamma as well as an increase in the secretion of tumor necrosis factor-alpha. Thus morphine altered the immune system by down-regulating splenocyte proliferation, because retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. We also evaluated the effects of joint murine retrovirus infection and protein undernutrition on the thymus cell subsets. Retrovirus infection was associated with a decrease in the absolute number of Thy 1+, CD4+ and CD8+ cells per thymus with the CD8+ cell subset being the most affected. Moreover, retrovirus-infected mice presented a dramatic decrease in the percentage of double-positive (CD4+ CD8+) cells in the thymus as well as changes in its immunoarchitecture. While protein undernutrition alone did not produce further differences between infected versus non-infected, protein-undernourished, morphine treatment induced a greater decrease in thymocyte number than that seen in retrovirus- or morphine-treated animals alone.

Vitamin E supplementation modulates cytokine production by thymocytes during murine AIDS

Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine AIDS, which is functionally similar to human AIDS. Retrovirus infection targeted the thymus, producing altered T cell differentiation via the dysregulation of thymocyte cytokine production. Human AIDS causes vitamin deficiencies, therefore the effects of dietary vitamin E supplementation were determined on the kinetics of cytokine production by concanavalin A-stimulated thymocytes in uninfected normal mice and mice with murine AIDS. Dietary supplementation, with a 15-fold increase in vitamin E (160 IU/1) in the liquid diet (National Research Council), modulated interleukin-2 (IL) production in both uninfected mice and retrovirus-infected mice. Vitamin E significantly reduced the level of IL-4 secretion in the uninfected mice at 4 and 8 weeks, but not at 12 and 16 weeks. It also significantly reduced IL-4 production, elevated by retrovirus infection. Vitamin E significantly reduced IL-6, and interferon-γ production increased in murine AIDS. The effects of dietary vitamin E on concanavalin A-induced proliferation of thymocytes were consistent with the finding of changes in IL-2 secretion. No effects of dietary vitamin E on thymus weight were observed in uninfected or retrovirus-infected mice, whereas vitamin E significantly increased serum and thymic vitamin E concentration, which had been reduced by retrovirus infection. These data indicate that dietary vitamin E supplementation can modulate cytokine production by thymocytes, affecting T cell differentiation, especially during retrovirus-induced immune dysfunction.